Published online February 3, 2004
Nucleic Acids Research, 2004, Vol. 32, No. 2 803±810
DOI: 10.1093/nar/gkh225
Recent horizontal intron transfer to a chloroplast
genome
Elena V. Sheveleva and Richard B. Hallick*
Department of Biochemistry and Molecular Biophysics, The University of Arizona, 1041 East Lowell Street,
Tucson, AZ 85721-0088, USA
Received September 22, 2003; Revised and Accepted December 18, 2003
ABSTRACT
Evidence is presented for the recent, horizontal
transfer of a self-splicing, homing group II intron
from a cyanobacteria to the chloroplast genome of
Euglena myxocylindracea. The psbA gene of
E.myxocylindracea was found to contain a single
2566 nt group II intron with a gene in domain 4 for a
575 amino acid maturase. The predicted secondary
structure and tertiary interactions of the group II
intron, as well as the derived maturase primary
sequence, most closely resemble the homing intron
of the cyanobacterium Calothrix and the rnl introns
of Porphyra purpurea mitochondria, while being
only distantly related to all other Euglena plastid
introns and maturases. All main functional domains
of the intron-encoded proteins of known homing
introns are conserved, including reverse transcriptase domains 1±7, the zinc ®nger domain and
domain X. The close relationship with cyanobacterial introns was con®rmed by phylogenetic
analysis. Both the full-length psbA intron and a
D-maturase variant self-splice in vitro in two independent assays. The psbA intron is the ®rst
example of a self-splicing chloroplast group II intron
from any organism. These results support the conclusion that the psbA intron is the result of a recent
horizontal transfer into the E.myxocylindracea
chloroplast genome from a cyanobacterial donor
and should prompt a reconsideration of horizontal
transfer mechanisms to account for the origin of
other chloroplast genetic elements.
INTRODUCTION
Group II introns are found in prokaryotic mRNA and in tRNA,
mRNA and rRNA of organelles in fungi, plants and protists.
Group II introns are ribozymes. They have a conserved
secondary structure, ®rst proposed by Michel as a central core
with six radiating helical domains, designated d1±d6 (1; see
review in 2). The catalytic center is formed by d1 and d5.
Several tertiary interactions are involved in the stabilization of
the catalytic core, including EBS1/IBS1, EBS2/IBS2, EBS3/
DDBJ/EMBL/GenBank accession no. AY290861
IBS3, a/a¢, b/b¢, e/e¢, d /d¢ and g/g¢. In addition to Watson±
Crick pairings, an 11 nt RNA motif z (AUGG ¼ CCUAA)
that is conserved in d1 of group II self-splicing introns
interacts with the domain V hairpin terminal loops with a
GNRA consensus sequence z¢. Similarly, in the h/h¢ interaction a GNRA hairpin loop that caps d6 interacts with another
11 nt motif within d2 (see review in 3).
Euglenoid chloroplast group II introns have the same core
secondary structure as other group II introns (4,5), con®rmed
by comparative analysis of cognate introns from two or more
euglenoid species (6,7). Euglenoid group II introns splice by
the same two-step mechanism as other group II introns (8).
Nevertheless, these introns comprise a class distinct from their
counterparts in other organelles and prokaryotes (see review in
9). Many are considerably shorter (252±671 nt) than typical
group II introns and lack some expected tertiary contacts.
More variability occurs in the catalytic d5 domain than in
more typical group II introns. Plastid genomes of all 17
Euglena spp. examined to date also contain a unique class of
chloroplast introns designated group III (9), a streamlined
derivative of group II introns. Group III introns have a narrow
size range of 73±119 nt, lack the group II intron-like d2±d5
domains and splice via a lariat intermediate utilizing the
unpaired adenosine (A*) within d6 (10). The 5¢-region of some
group III introns is somewhat similar to domain ID3 of group
II introns.
A small subset of group II introns, none of which are from
chloroplast genomes, have `homing' activity, the ability to
move horizontally into an intron-less allele of the same
genome or into a new genome. Subsequent intragenomic
homing could result in vertical evolution among direct
descendants of the ancestral genome. A detailed understanding of group II intron homing has come from studies of the
group II intron ai2 of yeast mitochondria and L1.Ltr B of
Lactococcus lactis (11,12). The L1.Ltr B intron RNA reverse
splices directly into the sense strand of a double-stranded
DNA target site, while the intron-encoded reverse transcriptase/maturase cleaves the antisense strand and uses it as primer
for reverse transcription of the inserted intron RNA (13±15).
The ability of group II introns to spread into ectopic sites has
also recently gained additional experimental support (16) and
is an attractive model for the introduction of spliceosomal
introns into the eukaryotic nucleus (16,17). Intron homing was
likely important for the proliferation of group II introns into
the genomes of bacteria and the organelle genomes of lower
eukaryotes and plants. There is little evolutionary evidence for
*To whom correspondence should be addressed. Tel: +1 520 621 3026; Fax: +1 520 621 1697; Email: [email protected]
Nucleic Acids Research, Vol. 32 No. 2 ã Oxford University Press 2004; all rights reserved
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Nucleic Acids Research, 2004, Vol. 32, No. 2
this process, as most group II introns in eukaryotes have lost
homing activity and an intron-encoded protein. The phylogenetic relationships and evolutionary history of group II
introns with reverse transcriptase genes have been reported
(3,5,18,19).
Although chloroplasts may have acquired group II introns
by horizontal transfer from a bacterial origin, none of the
current models envision recent evolutionary transfer into a
chloroplast genome. Furthermore, chloroplast intron-encoded
proteins (IEPs) are all evolutionarily distinct from their
counterparts in mobile introns and are thought to be the result
of vertical evolution. Plastid genomes originated by endosymbiosis of a cyanobacterium. After the discovery of introns
in cyanobacteria, these photosynthetic prokaryotes were
considered the source of introns in chloroplast genomes (20±
22). More important, horizontal gene transfer into chloroplast
genomes from other organisms was believed to be unlikely
(23±27). The acquisition of new introns and insertion into new
sites during vertical evolution could be explained by functional intragenomic mobility (homing) of group I and II
introns (28). There are contradictions to vertical chloroplast
evolution, such as the absence of chloroplast introns in some
algal chloroplast genomes considered the predecessors of
plant chloroplast genomes (29±31). In this study we present
evidence for recent transfer of a homing intron from a
cyanobacteria to the chloroplast genome of Euglena myxocylindracea. This result may exemplify a more generalized
ability of the chloroplast genomes of some species to acquire
other types of genetic information within plasmids, viruses
and transposable elements via horizontal DNA transfer.
MATERIALS AND METHODS
Euglena cultures and nucleic acid extraction
Euglena myxocylindracea (UTEX 1989) was obtained from
the University of Texas Culture Collection. Total nucleic acid
(TNA) extracts were prepared as previously described (32)
either directly from cultures obtained from the UTEX culture
collection grown on solid slants or following additional
growth in liquid culture. The RNA was isolated using Triazol
reagent (Gibco BRL) or double puri®ed using Ambion
Mini-plant puri®cation kits (Ambion Inc.).
PCR ampli®cation, cDNA synthesis and sequencing
Intron and intron-encoded maturase sequences were isolated
from E.myxocylindracea by PCR ampli®cation from primers
targeted to the psbA gene. A sample of 0.05±0.2 mg/ml of
nucleic acid extract (TNA) was ampli®ed from the synthetic
oligonucleotides P1 and P2. Primer P1 corresponds to
coordinates 559±581 (5¢-GGACCTTACCAGTTAATTGTATG) and primer P2 corresponds to coordinates 3598±3621
(5¢-AAGAAGAAATGTAAAGAACGAGAG) of the E.myxocylindracea psbA±psbK operons (accession no. AY290861).
Exon±intron boundaries were determined by RT±PCR followed by DNA sequencing with the same primers. Superscript
II (Invitrogen Inc.) was used for reverse transcription.
Recombinant plasmids
Plasmid PEYC 2009 contains 226 nt from the 5¢-exon, the
2566 nt group II intron containing mat4 and the ®rst 271 nt of
the 3¢-exon of psbA from E.myxocylindracea. The plastid
sequence was cloned distal to the phage T3 promoter in
pBSKS+ as an sst1±sst2 insert. PEYC 2010 contains the same
5¢-exon, 173 nt from the 3¢-exon and mat4 with a large internal
deletion leaving only 62 nt, also cloned behind the phage T3
promoter in pBSKS+.
In vitro transciption
In vitro radiolabeled transcripts were synthesized from 1 mg
linearized plasmids using transcription buffer, 3 mCi/ml
[a-32P]UTP (3000 Ci/mmol), 100 mM unlabeled UTP,
500 mM each of the other NTPs and MAXIscript T3 RNA
polymerase (Ambion Inc.). Transcripts were gel puri®ed on a
denaturing 4% polyacryalamide gel. RNA was excised and
soaked overnight at 4°C in 500 mM NH4Ac, 10 mM MgCl2,
0.1 mM EDTA, 0.1% SDS. To obtain non-radiolabeled
transcripts MEGAscript T3 (Ambion Inc.) was used. For
in vitro splicing reactions both labeled and non-labeled
transcripts were ethanol precipitated and then dissolved in
buffer containing 40 mM MOPS, 1 mM EDTA, 1 mM
dithiothreitol, 0.5 U/ml RNase inhibitor. Prior to splicing,
200 nM RNA was renatured by heating to 90°C for 1 min and
then immediately diluted into assay buffer at 30°C (33).
Self-splicing reactions in vitro
Transcripts were incubated for the indicated times at 42°C in
40 mM MOPS, 10 mM MgCl2, 1 mM dithiothreitol, 1 ml
RNase inhibitor and 500 mM (NH4)2SO4. Splicing products of
radioactive transcipts were analyzed on a denaturing 4%
polyacrylamide gel, followed by autoradiography. Splicing
products of non-radioactive transcripts were gel puri®ed and
analyzed by RT±PCR followed by DNA sequencing.
Computer analysis
Sequences similar to mat4 were identi®ed by BLAST searches
(34). Open reading frame analysis and determination of
putative amino acid sequences were done with the computer
program DNAStrider. Nucleotide and protein sequence
alignments were carried out using the PILEUP and Clustal
X programs (Genetics Computer Group Sequence Analysis
Package, version 8.0, Madison, WI). RNA folding was done
mostly manually and using the mFold program (35).
Phylogenetic analysis
The amino acid sequences of IEPs were aligned by PILEUP
and Clustal X. Phylogenetic trees on 19 amino acid sequences
were constructed using the neighbor joining and parsimony
analysis default settings of the program PAUP and two amino
acid sequences were taken as an outgroup (36). Bootstrap
values were from 1000 re-samplings. The following representatives were selected for analysis (gene name or locus tag is
followed by species name, with accession no. in parentheses).
Chloroplast genome: mat4, E.myxocylindracea (AY290861);
unnamed, Scenedesmus obliquus (P19593). Cyanobacteria:
ORF2, Calothrix sp. (CAA50529); alr8560, Nostos sp.
PCC 7120 (BAB77479); tlr0620, Thermosynechoccus
elongatus BP-1 (NP_681409); gll0177, Gloeobacter violaceus
(NP_923123); Tery0355, Trichodesmium erythraeum
(ZP_00071211); all5206, Nostoc sp. PCC 7120
(NP_489246). Other bacteria: Avin1823, Azotobacter vinelandii (ZP_00090141); pX01-07, Bacillus anthracis
Nucleic Acids Research, 2004, Vol. 32, No. 2
805
`euglenoid-type' group II introns. Rather, the predicted
secondary structure model and tertiary interactions conform
very closely to the Cal.x1 intron of Calothrix (22) and rnl
introns 1 and 2 of P.purpurea mitochondrial DNA (38).
The intron-encoded protein resembles cyanobacterial
reverse transcriptases
Figure 1. Schematic diagram of the psbA region. Group II introns are indicated by gray lollipops. (a) The E.gracilis psbA gene with four group II
introns. (b) The E.myxocylindracea psbA gene encoding a group II intron
with mat4.
(AAD32311.1); BC2646, Bacillus cereus ATCC 14579
(NP_832403); ORF34, Bacillus megaterium (NP_799494);
BT2297,
Bacteroides
thetaiotaomicron
VPI-5482
(NP_811210); L0272, Escherichia coli (AAC70140.1);
tlr0620, Xylella fastidiosa (AE003999, 11586±13295);
SAV226,
Streptomyces
avermitilis
(NP_821400);
Haso014801,
Haemophilus
somnus
(ZP_00131874).
Mitochondrial genome: ORF544, Porphyra purpurea
(NP_049293); ORF568, Pylaiella littoralis (NP_150379).
Bacterial group II intron outgroup: Clostridium dif®cile
(X98606); LtrA, Lactococcus lactis (U50902).
RESULTS
Identi®cation of a novel group II intron in the
E.myxocylindracea psbA gene
The psbA gene of Euglena gracilis was previously reported to
contain four `euglenoid' type group II introns. During a survey
of several additional Euglena spp. to determine if the
E.gracilis psbA introns are conserved, a single intron was
discovered in the chloroplast psbA gene of E.myxocylindracea
(Fig. 1). The intron insertion site, unique to E.myxocylindracea, is located within exon 3 in the corresponding
E.gracilis psbA gene. Six other Euglena spp. surveyed lack
both the four E.gracilis psbA introns and the novel
E.myxocylindracea intron. The intron and ¯anking coding
regions were ampli®ed by PCR, cloned and sequenced on both
strands (accession no. AY290861). The new intron is 2566 nt
in length. Encoded in the intron is an ORF of 1728 nt that
would encode a protein of 575 amino acids.
Structure of the intron
The E.myxocylindracea intron has typical group II intron
secondary structure with six helical domains (dI±dVI) radiating from a central core (Fig. 2). The intron-encoded protein
gene, located within dIV as expected, is designated mat4,
being the fourth example of a Euglena chloroplast intronencoded protein. All expected tertiary interactions between
introns and exons (EBS1/IBS1, EBS2/IBS2 and EBS3/IBS3)
and within the intron (a/a¢, b/b¢, e/e¢, d/d¢, g/g¢, z/z¢ and h/h¢)
were identi®ed (Fig. 2). The intron belongs to subgroup IIb
(37), with a putative d-d¢ pairing in dI and the EBS3 base A
paired with the ®rst nucleotide of the 3¢-exon. The new group
II intron is not similar to any other known Euglena chloroplast
The deduced amino acid sequence of mat4, when compared to
known proteins by BLAST analysis (34), was found to have E
values in the range 2 3 e±91 to e±126 for alignments with group
II intron-encoded and free standing reverse transcriptases from
cyanobacteria, as well as intron-encoded proteins of the
mitochondrial gene of the red alga P.purpurea. The highest
similarity of the E.myxocylindracea intron protein, with 43%
identity and 59±60% positives over the entire coding region, is
to a reverse transcriptase homolog from Nostoc sp. PCC 7120
which is encoded by the alr8560 gene (accession no.
NC_003273) and to a group II intron-encoded protein from
Calothrix (accession no. S40013). The Nostoc ORF (45989±
47791) is in d4 of a putative group II intron (45422±47910).
After cyanobacteria the highest similarity to mat4 was with
reverse transcriptase homologs ORF544 and ORF546 from
mitochondria of P.purpurea (35±37% identity and 55%
positives). Alignment scores between mat4 and the previously
described mat1±mat3 loci of Euglena chloroplasts and the
various matK loci of plant chloroplasts were very low
compared to the cyanobacterial reverse transcriptases, indicative of only a distant relationship to previously described
intron-encoded proteins of chloroplasts.
It is noteworthy that both the intron structure and the intronencoded protein gene are most similar to cyanobacterial and
red algal mitochondrial introns. This would be expected if the
intron resulted from horizontal transfer of a retrohoming
cyanobacterial intron.
The mat4 encoded protein
An amino acid sequence alignment of the mat4 IEP with IEPs
from P.purpurea and Calothrix is shown in Figure 3. All of the
main functional domains from P.purpurea and Calothrix are
conserved in E.myxocylindracea. These are the reverse
transcriptase, a Zn ®nger domain and domain X. Within the
reverse transcriptase domain are seven conserved amino acid
motifs (RT1±RT7) characteristic of retrotransposons and other
reverse transcriptases. The mat4 IEP sequence aligns with the
cyanobacterial and mitochondrial sequences in the Zn ®nger
domain, domain X and RT1±RT7 as well as they align with
each other. Additionally, the reverse transcriptase has an
additional domain RT0 which is characteristic of IEPs. One
functional motif in RT5 has the sequence YADD typical of
bacterial IEPs in retrohoming introns. Mutations in either Asp
residue cause the impairment of Mg2+ binding at the reverse
transcriptase active site and elimination of reverse transcriptase activity (11,39,40). A YADD motif is not present in other
maturases of euglenoids nor in maturases of other chloroplast
genomes.
The distance between domains RT4 and RT5 is slightly
longer in E.myxocylindracea compared to the other three
species. Domain X is a putative RNA-binding domain
associated with maturase activity (41). The Zn ®nger domain
is located distal to domain X of the protein and is associated
with intron mobility. It can be divided into N- and C-terminal
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Nucleic Acids Research, 2004, Vol. 32, No. 2
Figure 2. Primary sequence, secondary structure model and proposed tertiary interactions for the psbA intron of E.myxocylindracea. This model is based on
the predicted structure of group IIb introns proposed by Michel (47) and closely resembles the model for the Cal.x1 intron from the cyanobacteria
Calothrix (22).
regions. The N-terminal region is required for reverse splicing
into double-stranded DNA and DNA unwinding, while the
C-terminal region containing the conserved Zn ®nger-like and
endonuclease motifs is required for endonuclease activity
(42,43). The Zn ®nger domain is present in mat4, but absent in
all previously investigated maturases from euglenoids. Thus
the mat4 IEP is most closely related to cyanobacterial IEPs
and only distantly related to chloroplast IEPs.
mat4 from E.myxocylindracea could be expressed and
isolated from E.coli BL21(DE3) (data not shown), although
apparently in an inactive form. This is the ®rst maturase from
euglenoids which we have been able to express in E.coli cells,
perhaps a consequence of a recent prokaryotic origin.
Self-splicing in vitro of the intron containing mat4
The ability of the psbA group II intron and a D-IEP variant to
undergo self-splicing in vitro was investigated. In vitro RNA
transcripts were incubated at 42°C in 0.5 M ammonium sulfate
for 0±60 min. The resulting RNA products were ampli®ed by
RT±PCR and resolved by agarose gel electrophoresis
(Fig. 4A). A product corresponding in size to spliced exons
is detected following a 10±60 min incubation. Lowering the
temperature or ionic strength or substituting ammonium
chloride all resulted in decreased spliced product formation.
The RT±PCR products of the D-IEP psbA pre-mRNA are
shown in Figure 4B. The ampli®ed product from the spliced
psbA mRNA is the major species after either 20 or 60 min of
incubation. The products labeled E5¢±E3¢ were con®rmed to
represent correctly spliced psbA mRNA exons by cloning and
DNA sequence analysis (data not shown). The in vitro selfsplicing reaction was repeated with 32P-labeled psbA premRNA (Fig. 4C). Products corresponding to both the spliced
exons and the excised intron lariat were obtained. The excised
intron lariat is evident after 20 and 60 min incubation. Thus,
Nucleic Acids Research, 2004, Vol. 32, No. 2
807
Figure 3. Alignment of group II intronic IEP. Species selected are: C.sp., Calothrix sp., ORF2 (X71404); P.p., P.purpurea, ORF544 (NC_002007.1); E.m.,
E.myxocylindracea, mat4 (AY290861). The highlighted domains are reverse transcriptase motifs 0±7 (RT0±RT7), RNA maturase (X, required for splicing)
and Zn ®nger domain, required for mobility. Conserved amino acids are indicated by red, similar amino acids are indicated by blue. Similar amino acids are
de®ned as: E and D; F and Y; R and K; L, V, I and M; S and T. A yellow background indicates reverse transcriptase (RT) domains, blue maturase (X) domain
and orange the Zn ®nger domain.
by two different assays, the E.myxocylindracea psbA intron
was shown to self-splice in vitro. This is the ®rst example of a
self-splicing group II intron from any chloroplast genome.
Phylogenetic analysis of mat4
To investigate the evolutionary relationship between mat4 and
related intron-encoded proteins and reverse transcriptase
proteins, a phylogenetic analysis was performed on proteins
of cyanobacterial, bacterial, mitochondrial and chloroplast
origin using neighbor joining and parsimony analysis (36).
Sequences for evolutionary comparisons were chosen from
among the 50 highest BLAST similarity scores. Multiple
sequences from the same species and sequences which did not
have all domains present in mat4, with the exception of Nostoc
sp., were omitted. The only chloroplast genome sequence
represented, other than mat4, was a reverse transcriptase from
S.obliquus. Other euglenoid maturases and maturases from
chloroplast genomes of charophytes and plants were also too
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Nucleic Acids Research, 2004, Vol. 32, No. 2
Figure 4. In vitro splicing. Splicing products of non-radioactive transcripts
were puri®ed and a RT±PCR was carried out: (A) plasmid PEYC2009; (B)
plasmid PEYC2010). Splicing products of radioactive transcripts were
analyzed on a denaturing 4% polyacrylamide gel, which was dried, put on
X-ray ®lm and developed the next day (C). M, marker l DNA±BstEII;
C, control.
divergent for this analysis. Some reverse transcriptases
included are in the chloroplast-type reverse transcriptase
group de®ned by Zimmerly et al. (19). Two bacterial reverse
transcriptases of mitochondrial type were chosen as an
outgroup. The resulting 19 sequences were from cyanobacteria, proteobacteria and mitochondrial genomes. The
results of the evolutionary analysis are shown in Figure 5.
Both mat4 and the reverse transcriptase from the mitochondrial genome of P.purpurea are deeply rooted in the
cyanobacterial clade. One of two bacterial clades includes
representative sequences from cyanobacteria as well as the
mitochondria genome of brown algae. The most closely
related chloroplast sequence to mat4 is a reverse transcriptase
from S.obliquus, which is the most distantly related representative of a clade with a mitochondrial protein from Pylaiella
and several bacteria sequences. From this phylogenetic
analysis, mat4 is more closely related to cyanobacteria
sequences than reverse transcriptase from the green algae
S.obliquus or any other plastid maturase.
DISCUSSION
The structural similarities of the psbA intron RNA, the in vitro
self-splicing activity and the phylogenetic position of the IEP
within a cyanobacterial clade support the conclusion that the
psbA intron is the result of a recent horizontal transfer into the
E.myxocylindracea chloroplast genome from a cyanobacterial
donor. Previous models for the evolution of chloroplast
Figure 5. Phylogenetic trees on 21 amino acid sequences including two outgroup sequences were constructed using the distance (neighbor joining, NJ)
method of the program PAUP. Bootstrap values from 1000 re-samplings are given at the nodes of the tree. Branch lengths are proportional to the expected
mean number of substitutions per site along the branch, as quanti®ed by the scale bar. Parsimony analysis had similar branching as in the NJ tree (data not
shown). chl., chloroplast genome; mit., mitochondrial genome. Bacteria include bacteria other than cyanobacteria.
Nucleic Acids Research, 2004, Vol. 32, No. 2
genetic elements have emphasized a common origin during
the primary endosymbiotic event that gave rise to chloroplast
genomes. The absence of introns in chloroplast genomes of the
early branching algal species, but their reappearance in green
algal chloroplast genomes and their descendants, including the
vascular plants, has been explained by loss of introns in the
chloroplast genomes of early branching species (29±31). The
variation in intron content among plastids from different
sources was attributed to vertical transfer subsequent to
primary endosymbiosis. There is now evidence for an
alternative pathway of chloroplast genome evolution. Introns
in chloroplasts may have originated either from the primary
endosymbiont or due to secondary horizontal transfer(s) from
a cyanobacterial donor. Given the variety in chloroplast intron
location and structure, horizontal transfer may have occurred
many times in different plastid lineages, most notably in the
euglenoid protists.
Euglena plastid genomes are unique with respect to
chloroplast introns, due to the large numbers, structural
diversity, multitude of IEPs and high degree of degeneracy.
The discovery of a new psbA intron with strictly cyanobacterial features suggests an attractive explanation for intron
history in euglenoid plastids. Multiple horizontal intron
transfer events from cyanobacteria into plastid genomes in
this lineage may have given rise to different populations of
chloroplast introns. Following acquisition of a foreign intron,
intragenomic mobility could account for the spread of introns
to new target sites, including into existing introns, accounting
for the formation of a variety of classes of twintrons (8). One
cyanobacterial intron may have evolved into group III introns,
while other cyanobacterial introns evolved into different
varieties of group II introns. Support for this model lies in the
variety of Euglena chloroplast IEPs (`maturases') described to
date, including mat1 speci®c for a group III twintron of psbC,
mat2 speci®c for a group II intron of psbC, mat3 within a
group II intron of psbD, mat4 described in this report and mat5
within a group II intron of psbA in Euglena spirogyra
(E.Sheveleva and R.B.Hallick, unpublished observations).
Several factors may have in¯uenced the evolution of introns in
plastid genomes. An early event may be the loss of
intragenomic mobility, caused by mutation of domains in
the IEPs. Additional streamlining of both the IEPs and the
RNA is likely the consequence of host factors assuming part of
the splicing reaction. The ultimate loss of IEPs in most plastid
introns re¯ects a major role of the host in catalyzing the
splicing reactions. Another evolutionary factor is the type of
gene where introns are inserted, as evolution of introns follows
the evolution of the genes in which they are located. Group III
introns are more common in non-photosynthetic genes,
whereas group II introns are more common in photosynthesis-related genes (9). Finally, different classes of plastid
introns must also re¯ect the evolutionary time since a
horizontal transfer event. The most deeply rooted introns,
such as the group III twintron with the IEP mat1 in psbC of
many euglenoid species, were presumably acquired very early
compared to the recent arrival of the cyanobacterial-like psbA
intron of this report.
The formation of algal chloroplast genomes occurred more
than 1.2 billion years ago. Primary plastids of red and green
algae have spread laterally among distantly related organisms
through the process of secondary endosymbiosis, in which a
809
heterotrophic eukaryote, including many euglenoids, retains
the photosynthetic apparatus of algae (44). Green algae are the
closest predecessors of euglenoids. The possibility of horizontal gene transfer into chloroplast genomes has been
discussed by others, but either not proven or considered
unlikely (23±27). There is precedent for the concept of
horizontal intron transfer between organisms. Horizontal
intron and gene transfer are well known in bacteria (reviewed
in 19,45). Likewise, the appearance of cyanobacterial-like
introns in the mitochondrial genome of red and brown alga are
presumably results of horizontal gene transfer (19,38,46).
The discovery of horizontal intron transfer in Euglena
should prompt a reconsideration of horizontal transfer of
introns and other genetic elements in the evolutionary history
of chloroplast genomes. The differences in intron and gene
content among chloroplast genomes of different species could
re¯ect both differential gene loss from the primary endosymbiont and also horizontal transfer of new genes to some plastid
lineages. Many contemporary plastid genomes may be an
evolutionary mosaic of genes from the primary endosymbiotic
event that gave rise to all plastid genomes and other genes
acquired through secondary horizontal transfer events from
many different sources.
ACKNOWLEDGEMENTS
We would like to thank Jane Dugas for technical assistance
with the manuscript. We would also like to thank Dr W. Birky
for assistance with the phylogenetic analysis. This work was
supported by NIH grant GM35665.
REFERENCES
1. Michel,F., Jacquier,A. and Dujon,B. (1982) Comparison of fungal
mitochondrial introns reveals extensive homologies in RNA secondary
structure. Biochimie, 64, 867±881.
2. Michel,F. and Ferat,J.L. (1995) Structure and activities of group II
introns. Annu. Rev. Biochem., 64, 435±461.
3. Bonen,L. and Vogel,J. (2001) The ins and outs of group II introns. Trends
Genet., 17, 322±331.
4. Keller,M. and Michel,F. (1985) The introns of the Euglena gracilis
chloroplast gene which codes for the 32-kDa protein of photosystem II.
FEBS Lett., 179, 69±73.
5. Michel,F., Umesono,K. and Ozeki,H. (1989) Comparative and functional
anatomy of group II catalytic intronsÐa review. Gene, 82, 5±30.
6. Thompson,M.D., Copertino,D.W., Thompson,E., Favreau,M.R. and
Hallick,R.B. (1995) Evidence for the late origin of introns in chloroplast
genes from an evolutionary analysis of the genus Euglena. Nucleic Acids
Res., 23, 4745±4752.
7. Thompson,M.D., Zhang,L., Hong,L. and Hallick,R.B. (1997) Extensive
structural conservation exists among several homologs of two Euglena
chloroplast group II introns. Mol. Gen. Genet., 257, 45±54.
8. Copertino,D.W. and Hallick,R.B. (1991) Group II twintron: an intron
within an intron in a chloroplast cytochrome b-559 gene. EMBO J., 10,
433±442.
9. Copertino,D.W. and Hallick,R.B. (1993) Group II and group III introns
of twintrons: potential relationships with nuclear pre-mRNA introns.
Trends Biochem. Sci., 18, 467±471.
10. Copertino,D.W., Hall,E.T., Van Hook,F.W., Jenkins,K.P. and
Hallick,R.B. (1994) A group III twintron encoding a maturase-like gene
excises through lariat intermediates. Nucleic Acids Res., 22, 1029±1036.
11. Moran,J.V., Zimmerly,S., Eskes,R., Kennell,J.C., Lambowitz,A.M.,
Butow,R.A. and Perlman,P.S. (1995) Mobile group II introns of yeast
mitochondrial DNA are novel site-speci®c retroelements. Mol. Cell.
Biol., 15, 2828±2838.
12. Matsuura,M., Saldanha,R., Ma,H., Wank,H., Yang,J., Mohr,G.,
Cavanagh,S., Dunny,G.M., Belfort,M. and Lambowitz,A.M. (1997)
810
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
Nucleic Acids Research, 2004, Vol. 32, No. 2
A bacterial group II intron encoding reverse transcriptase, maturase and
DNA endonuclease activities: biochemical demonstration of maturase
activity and insertion of new genetic information within the intron. Genes
Dev., 11, 2910±2924.
Zimmerly,S., Guo,H., Perlman,P.S. and Lambowitz,A.M. (1995) Group
II intron mobility occurs by target DNA-primed reverse transcription.
Cell, 82, 545±554.
Cousineau,B., Smith,D., Lawrence-Cavanagh,S., Mueller,J.E., Yang,J.,
Mills,D., Manias,D., Dunny,G., Lambowitz,A.M. and Belfort,M. (1998)
Retrohoming of a bacterial group II intron: mobility via complete reverse
splicing, independent of homologous DNA recombination. Cell, 94,
451±462.
Eskes,R., Liu,L., Ma,H., Chao,M.Y., Dickson,L., Lambowitz,A.M. and
Perlman,P.S. (2000) Multiple homing pathways used by yeast
mitochondrial group II introns. Mol. Cell. Biol., 20, 8432±8446.
Cousineau,B., Lawrence,S., Smith,D. and Belfort,M. (2000)
Retrotransposition of a bacterial group II intron. Nature, 404, 1018±1021.
Eickbush,T.H. (2000) Molecular biology. Introns gain ground. Nature,
404, 940±943.
Toro,N., Molina-Sanchez,M.D. and Fernandez-Lopez,M. (2002)
Identi®cation and characterization of bacterial class E group II introns.
Gene, 299, 245±250.
Zimmerly,S., Hausner,G. and Wu,X. (2001) Phylogenetic relationships
among group II intron ORFs. Nucleic Acids Res., 29, 1238±1250.
Xu,M.Q., Kathe,S.D., Goodrich-Blair,H., Nierzwicki-Bauer,S.A. and
Shub,D.A. (1990) Bacterial origin of a chloroplast intron: conserved selfsplicing group I introns in cyanobacteria. Science, 250, 1566±1570.
Kuhsel,M.G., Strickland,R. and Palmer,J.D. (1990) An ancient group I
intron shared by eubacteria and chloroplasts. Science, 250, 1570±1573.
Ferat,J.L. and Michel,F. (1993) Group II self-splicing introns in bacteria.
Nature, 364, 358±361.
Martin,W., Stoebe,B., Goremykin,V., Hapsmann,S., Hasegawa,M. and
Kowallik,K.V. (1998) Gene transfer to the nucleus and the evolution of
chloroplasts. Nature, 393, 162±165.
Martin,W. and Schnarrenberger,C. (1997) The evolution of the Calvin
cycle from prokaryotic to eukaryotic chromosomes: a case study of
functional redundancy in ancient pathways through endosymbiosis.
Curr. Genet., 32, 1±18.
Adachi,J., Waddell,P.J., Martin,W. and Hasegawa,M. (2000) Plastid
genome phylogeny and a model of amino acid substitution for proteins
encoded by chloroplast DNA. J. Mol. Evol., 50, 348±358.
Vogl,C., Badger,J., Kearney,P., Li,M., Clegg,M. and Jiang,T. (2003)
Probabilistic analysis indicates discordant gene trees in chloroplast
evolution. J. Mol. Evol., 56, 330±340.
Delwiche,C.F. and Palmer,J.D. (1996) Rampant horizontal transfer and
duplication of rubisco genes in eubacteria and plastids. Mol. Biol. Evol.,
13, 873±882.
Yang,J., Zimmerly,S., Perlman,P.S. and Lambowitz,A.M. (1996)
Ef®cient integration of an intron RNA into double-stranded DNA by
reverse splicing. Nature, 381, 332±335.
Lemieux,C., Otis,C. and Turmel,M. (2000) Ancestral chloroplast genome
in Mesostigma viride reveals an early branch of green plant evolution.
Nature, 403, 649±652.
Turmel,M., Otis,C. and Lemieux,C. (1999) The complete chloroplast
DNA sequence of the green alga Nephroselmis olivacea: insights into the
architecture of ancestral chloroplast genomes. Proc. Natl Acad. Sci. USA,
96, 10248±10253.
31. Douglas,S.E. and Penny,S.L. (1999) The plastid genome of the
cryptophyte alga, Guillardia theta: complete sequence and conserved
synteny groups con®rm its common ancestry with red algae. J. Mol.
Evol., 48, 236±244.
32. Thompson,M.D., Copertino,D.W., Thompson,E., Favreau,M.R. and
Hallick,R.B. (1995) Evidence for the late origin of introns in chloroplast
genes from an evolutionary analysis of the genus Euglena. Nucleic Acids
Res., 23, 4745±4752.
33. Saldanha,R., Chen,B., Wank,H., Matsuura,M., Edwards,J. and
Lambowitz,A.M. (1999) RNA and protein catalysis in group II intron
splicing and mobility reactions using puri®ed components. Biochemistry,
38, 9069±9083.
34. Altschul,S.F., Gish,W., Miller,W., Myers,E.W. and Lipman,D.J. (1990)
Basic local alignment search tool. J. Mol. Biol., 215, 403±410.
35. Zuker,M., Jaeger,J.A. and Turner,D.H. (1991) A comparison of optimal
and suboptimal RNA secondary structures predicted by free energy
minimization with structures determined by phylogenetic comparison.
Nucleic Acids Res., 19, 2707±2714.
36. Swofford,D.L. (2003) Phylogenetic Analysis Using Parsimony (*and
Other Methods), Version 4. Sinauer Associates, Sunderland, MA.
37. Costa,M., Michel,F. and Westhof,E. (2000) A three-dimensional
perspective on exon binding by a group II self-splicing intron. EMBO J.,
19, 5007±5018.
38. Burger,G., Saint-Louis,D., Gray,M.W. and Lang,B.F. (1999) Complete
sequence of the mitochondrial DNA of the red alga Porphyra purpurea.
Cyanobacterial introns and shared ancestry of red and green algae. Plant
Cell, 11, 1675±1694.
39. Jacobo-Molina,A., Clark,A.D.,Jr, Williams,R.L., Nanni,R.G., Clark,P.,
Ferris,A.L., Hughes,S.H. and Arnold,E. (1991) Crystals of a ternary
complex of human immunode®ciency virus type 1 reverse transcriptase
with a monoclonal antibody Fab fragment and double-stranded DNA
Ê resolution. Proc. Natl Acad. Sci. USA, 88,
diffract X-rays to 3.5-A
10895±10899.
40. Jacobo-Molina,A., Ding,J., Nanni,R.G., Clark,A.D.,Jr, Lu,X., Tantillo,C.,
Williams,R.L., Kamer,G., Ferris,A.L., Clark,P. et al. (1993) Crystal
structure of human immunode®ciency virus type 1 reverse transcriptase
Ê resolution shows bent
complexed with double-stranded DNA at 3.0 A
DNA. Proc. Natl Acad. Sci. USA, 90, 6320±6324.
41. Mohr,G., Perlman,P.S. and Lambowitz,A.M. (1993) Evolutionary
relationships among group II intron-encoded proteins and identi®cation
of a conserved domain that may be related to maturase function. Nucleic
Acids Res., 21, 4991±4997.
42. Doolittle,R.F., Feng,D.F., Johnson,M.S. and McClure,M.A. (1989)
Origins and evolutionary relationships of retroviruses. Q. Rev. Biol., 64,
1±30.
43. McClure,M.A. (1991) Evolution of retroposons by acquisition or deletion
of retrovirus-like genes. Mol. Biol. Evol., 8, 835±856.
44. McFadden,G.I. (2001) Chloroplast origin and integration. Plant Physiol.,
125, 50±53.
45. Ochman,H. (2001) Lateral and oblique gene transfer. Curr. Opin. Genet.
Dev., 11, 616±619.
46. Fontaine,J.M., Goux,D., Kloareg,B. and Loiseaux-de Goer,S. (1997) The
reverse-transcriptase-like proteins encoded by group II introns in the
mitochondrial genome of the brown alga Pylaiella littoralis belong to
two different lineages which apparently coevolved with the group II
ribosyme lineages. J. Mol. Evol., 44, 33±42.
47. Michel,F. and Ferat,J.L. (1995) Structure and activities of group II
introns. Annu. Rev. Biochem., 64, 435±461.